- Record: found
- Abstract: found
- Article: found
Cardiac tamponade in medical patients: a 10-year follow-up survey.
Author(s):
Jean-Christophe Cornily
1 ,
Pierre-Yves Pennec ,
Philippe Castellant ,
Eric Bezon ,
Grégoire Le Gal ,
Martine Gilard ,
Yannick Jobic ,
Jacques Boschat ,
Jean-Jacques Blanc
Publication date (Electronic):
2008
Journal:
Cardiology
Publisher:
S. Karger AG
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Cardiac tamponade represents a life-threatening condition that may complicate almost any cause of pericarditis. We conducted a 10-year prospective survey on patients with cardiac tamponade requiring an emergency drainage.
Related collections
Most cited references13
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- Article: not found
Tuberculous pericarditis.
The incidence of tuberculous pericarditis is increasing in Africa as a result of the human immunodeficiency virus (HIV) epidemic. The primary objective of this article was to review and summarize the literature on the pathogenesis, diagnosis, and management of tuberculous pericarditis. We searched MEDLINE (January 1966 to May 2005) and the Cochrane Library (Issue 1, 2005) for information on relevant references. A "definite" diagnosis of tuberculous pericarditis is based on the demonstration of tubercle bacilli in pericardial fluid or on a histological section of the pericardium; "probable" tuberculous pericarditis is based on the proof of tuberculosis elsewhere in a patient with otherwise unexplained pericarditis, a lymphocytic pericardial exudate with elevated adenosine deaminase levels, and/or appropriate response to a trial of antituberculosis chemotherapy. Treatment consists of the standard 4-drug antituberculosis regimen for 6 months. It is uncertain whether adjunctive corticosteroids are effective in reducing mortality or progression to constriction. Surgical resection of the pericardium remains the appropriate treatment for constrictive pericarditis. The timing of surgical intervention is controversial, but many experts recommend a trial of medical therapy for noncalcific pericardial constriction, and pericardiectomy in nonresponders after 4 to 8 weeks of antituberculosis chemotherapy. Research is needed to improve the diagnosis, assess the effectiveness of adjunctive steroids, and determine the impact of HIV infection on the outcome of tuberculous pericarditis.
- Record: found
- Abstract: found
- Article: not found
Genetic Variation in OAS1 Is a Risk Factor for Initial Infection with West Nile Virus in Man
Jean Lim, Andrea Lisco, David McDermott … (2009)
Introduction West Nile virus (WNV) is a re-emerging flavivirus transmitted by mosquitoes to several species of birds. Humans and several other mammalian species may be accidental dead-end hosts. First isolated in Uganda from a febrile woman in 1937 [1], WNV has caused sporadic outbreaks in the Middle East, Africa, Western Asia, Europe, and Australia. In the Western Hemisphere, it was first isolated from a patient during an outbreak of meningoencephalitis in New York in 1999 [2],[3],[4]. Since then it has rapidly spread across the United States into Canada and Central and South America, and has caused annual outbreaks of disease. Through November 18, 2008, there have been 28,906 US laboratory-confirmed symptomatic WNV-seropositive cases reported to the Centers for Disease Control and Prevention (CDC), with 1121 (3.9%) WNV-induced deaths (www.cdc.gov). In addition to human infections, the virus has caused significant morbidity and mortality in birds and horses. In the US, seroprevalence is ∼3% in the general population and 20–30% of infected individuals have been estimated to become symptomatic [5]. Clinical manifestations include West Nile fever (WNF) and WNV-induced neuroinvasive disease (WNND), which manifests primarily as meningitis and encephalitis [6]. To date, no specific antiviral agents or vaccines have been approved by the FDA for human WNV infection, and treatment is supportive. Survivors of WNND may develop long-term neurologic sequelae. Viral infections in man are typically controlled in part by early induction of type I interferon (IFN), which initiates a cascade of innate mechanisms that impair virtually all aspects of the viral life cycle. Genetic studies in mice have identified several type I IFN-regulated anti-viral effector pathways that control WNV replication, including the double-stranded RNA-dependent protein kinase (PKR) mechanism of protein synthesis inhibition, and the 2′–5′ oligoadenylate synthetase (OAS) pathway of RNaseL-mediated RNA degradation [7]. OAS enzymes catalyze the synthesis of 2′–5′-linked oligoadenylates (2–5A) from ATP, which can then bind and activate latent RNaseL resulting in the degradation of host and viral RNAs [8]. Targeted gene disruption in mice has revealed a critical role for the type I IFN Receptor, PKR and RNaseL for survival following WNV infection [7],[9]. A critical role for OAS emerged from genetic analysis of the mouse locus flv, which determines in an autosomal dominant manner susceptibility to WNV and other flaviviruses [10],[11]. Flv has recently been mapped by two groups working independently [12],[13] to a missense mutation in the 2′–5′ oligoadenylate synthetase 1b (oas1b) gene, which results in a truncated protein. How this mutation actually works has not yet been fully delineated. It clearly is not redundant with the multiple other oas1 genes found in the mouse genome [14]. However, there is evidence that protection may not actually be mediated through RNase L [15], and it is not yet known whether the oas1b gene product is enzymatically active [16]. The genetic data suggesting a role for oas1b in protection against WNV in mice are supported by in vitro experiments showing that WNV replication in cells expressing wild-type oas1b is less efficient than in cells expressing the truncated form [17],[18]. Furthermore, genetic knock-in of the resistant oas1b allele into a susceptible mouse strain resulted in resistance to Yellow Fever Virus, a related flavivirus [19]. WNV susceptibility has not been reported in this knock-in mouse. Taken together, the combined in vivo and in vitro data suggest an important role of oas1b in innate resistance to flavivirus infection in the mouse. Thus, we hypothesized that polymorphisms in the human ortholog of oas1b, OAS1 [14], could influence outcome in humans exposed to WNV. Established risk factors for human WNV disease include age, immunosuppression, and genetic deficiency in the chemokine receptor/HIV-1 coreceptor CCR5, due to homozygous inheritance of the defective allele CCR5Δ32 [20],[21]. Despite the strong epidemiologic association with CCR5Δ32, only ∼4% of Caucasian symptomatic WNV-infected individuals can be accounted for by CCR5 deficiency, indicating that other genetic risk factors for WNV disease may exist. A small case-control study (n = 27 cases) failed to find evidence of association of severe WNV disease with two OAS1 SNPs [22]. Instead, the investigators found a strong association with a silent variant (rs3213545) in OAS-Like (OASL), a paralogue of OAS1 located approximately 8 megabases away that has not been found to encode a protein with OAS activity [8]. In this report, we attempted to validate the OASL association with WNV, but could not. Instead, we found a consistent association of an OAS1 SNP and infection with WNV from samples collected from five independent US centers, and with WNV replication in human lymphoid tissue ex vivo. Results Structure and function of OAS1 SNP rs10774671 To investigate the role of the OAS system in controlling outcome after WNV exposure in man, we scanned the OAS1 gene for known polymorphisms that occur in this region. Unlike the mouse genome, where 8 copies of oas1 have been identified (oas1a-h), there is only one copy of OAS1 in the human genome, although alternative splicing gives rise to several isoforms [14]. All OAS1 splice variants include exons 1–5, but vary with regard to downstream exons, and thus produce proteins of various sizes, including the p42, p44, p46, p48, and p52 forms [14],[23]. We identified common SNPs in the OAS1 gene region in the CEPH cohort using data from the International HapMap Project (www.hapmap.org). Human OAS1 is one of three genes in the OAS cluster, ordered OAS1, OAS3 and OAS2 on chromosome 12 [24]. As shown in Figure 1, at least eleven SNPs in the OAS1 region have been found to cluster into two blocks of linkage disequilibrium (LD). The first block contains four SNPs, three of which are found within the first intron (rs7956880, rs10744785, and rs4766662) and one of which (rs2158390) is found in the 5′UTR. The second block contains seven SNPs, with rs3741981 in exon 3; rs2057778, rs2285934 and rs2285934 in intron 3; rs10774671 located at the last nucleotide in intron 5; rs1051042 and rs2660 in exon 7; and rs7135577 in the 3′UTR. The SNPs found in block 1 are not in coding regions. The six SNPs in block 2 excluding rs3741981 are in tight LD (Figure 1, the black region in block 2) and form two haplotypes that have been previously shown by Bonnevie-Nielsen and colleagues to be highly associated with the level of OAS enzymatic activity measured in peripheral blood mononuclear cells [23]. These investigators concluded that rs10774671 is most likely to be the functional SNP in this block because it sits at the last nucleotide of intron 5 in the OAS1 gene and serves as a splice acceptor site for exon 7. The G allele is predicted to allow splicing to occur resulting in the production of a p46 form with high enzymatic OAS activity. The A allele is predicted to prevent splicing at this site; instead, splicing occurs further downstream, resulting in two other forms, designated p48 and p52 associated with lower OAS enzymatic activity. Heterozygotes were reported to have intermediate OAS activity [23]. Although other splice variants of this gene exist, including p42 and p44, the splice variants that arise from the use of exon 7 (p46, p48, and p52) are controlled by rs10774671 [23],[24]. Thus, this polymorphism is a biologically plausible genetic probe to investigate the role of OAS1 in human WNV disease. 10.1371/journal.ppat.1000321.g001 Figure 1 Haplotype block structure of the human OAS1 gene in the CEPH cohort. The location of rs10774671 (boxed) and the surrounding SNPs in and around the OAS1 gene are shown. LD block structure of the OAS1 gene found in the CEPH cohort was analyzed using Haploview version 4.0. Pairwise r2 values are scaled in gray with higher r2 values shown in darker color; black indicates an r2 = 1. Study participants and genotype analysis of OAS1 SNP rs10774671 To test whether this SNP is associated with WNV infection, we obtained serum or plasma from five independent US sources of Caucasian WNV+ individuals (n = 501). We collected symptomatic WNV-seropositive patient samples (n = 331) from the following state public health departments and outbreak years: Arizona (2004), California (2005), Colorado, (2003) and Illinois (2005–6) as detailed in Table 1. These samples were from individuals who sought medical attention and where WNV was considered in the differential diagnosis because of compatible signs and symptoms (fever, meningitis, encephalitis) and confirmed by serologic testing. We also obtained asymptomatic WNV-seropositive blood donor samples (n = 170) from the American Red Cross (ARC) national blood supply screening program, identified as WNV nucleic acid amplification test (NAT) reactive and confirmed by serologic testing. These individuals remained symptom-free, according to self-assessment in a follow-up questionnaire, for at least 2 weeks post-donation [25],[26]. 10.1371/journal.ppat.1000321.t001 Table 1 Characteristics and genotypes of self-reporting Caucasian study subjects. AZ WNV+ CO WNV+ CA WNV+ IL WNV+ ARC WNV+ Combined WNV+ ARC WNV− RBD WNV− Combined WNV− Number 135 72 87 37 170 501 192 360 552 Mean age (y) 57±18 50±18 54±18 54±19 52±15 53±17 49±15 55±13 % Male sex 52.6 43.1 51.7 62.2 72.9 58.7 56.3 58.3 56.8 % WNFa 36.3 22.2 62.1 – 0 36 – – – % WNNDa 60 77.8 31 – 0 49.5 – – – % Unspecifieda 3.7 0 6.9 100 0 14.5 – – – % Mortality (n) 4.4 (6) 1.4 (1) 6.9 (6) – 0 4.4 (13)b – – – % OAS1 AA (n) 51.1 (69) 48.6 (35) 47.1 (41) 51.4 (19) 48.8 (83) 49.3 (247) 40.6 (78) 36.7 (132) 38.0 (210) % OAS1 AG (n) 38.5 (52) 44.4 (32) 35.7 (31) 48.6 (18) 38.2 (65) 39.5 (198) 46.2 (89) 46.4 (167) 46.4 (256) % OAS1 GG (n) 10.4 (14) 6.9 (5) 17.2 (15) 0.0 (0) 12.9 (22) 11.2 (56) 13.0 (25) 16.9 (61) 15.6 (86) % OAS-Like TT (n) 6.7 (9) 15.3 (11) 6.9 (6) 10.8 (4) 10.6 (18) 9.6 (48) 12.0 (23) 8.9 (32) 10.0 (59) % OAS-Like CT (n) 44.4 (60) 44.4 (32) 39.1 (34) 56.8 (21) 40.0 (68) 42.9 (215) 41.7 (80) 41.9 (151) 41.8 (245) % OAS-Like CC (n) 48.9 (66) 40.3 (29) 54.0 (47) 32.4 (12) 49.4 (84) 47.5 (238) 46.4 (89) 49.2 (177) 48.2 (281) a Cases from US states were classified into either West Nile fever (WNF), West Nile neuroinvasive disease (WNND), or unspecified disease categories by physician interview. All samples from ARC experienced no symptoms as assessed by questionnaire ≥2 weeks post-donation. b Mortality data do not include Illinois for which clinical outcome was not provided. RBD, random blood donors; AZ, Arizona; CO, Colorado; CA, California; IL, Illinois; ARC, American Red Cross; WNV+, WNV-seropositive individuals; WNV−, WNV-seronegative controls. All groups were in Hardy-Weinberg equilibrium. Genotypic frequencies where the total does not equal 100.0% are due to rounding imprecision. For comparison, we obtained two groups of US Caucasian control samples (n = 552). The first was comprised of healthy US Caucasian random blood donors (RBD) collected prior to the introduction of WNV into the US in 1999 (n = 360). The second consisted of healthy US blood donors collected by the ARC who were identified during routine blood screening as WNV false positives (initial reactivity by WNV NAT that could not be replicated and WNV-seronegative (n = 192)). We limited the analysis to self-reporting Caucasian individuals because the allele frequency of the OAS1 rs10774671 SNP varies according to race (www.ncbi.nlm.nih.gov/projects/SNP/). In both cases and controls, the overall genotyping success rate was >96.0%. 156 patient samples were re-tested using a second genotyping method with 100% concordance. Within each group (WNV+ and control), the OAS1 genotypes were in Hardy-Weinberg equilibrium (HWE; p = 0.51 and p = 0.93, respectively). In our control samples (n = 552), the frequency of the OAS1 rs10774671 AA genotype shown in Table 1 (38.0%) was consistent with the observed AA genotypic frequency in other published Caucasian cohorts representing over 5000 individuals predominantly from Europe (36.8–41.1%) [27],[28],[29] as well as the published report in the CEPH cohort in the HapMap Project (39%, n = 59; www.hapmap.org). OAS1 rs10774671 AA genotype is elevated in WNV-infected individuals A genotypic contingency analysis (3×2) for OAS1 rs10774671 revealed a statistically significant association when WNV-positive samples were compared to controls (p = 0.0008). In our analysis, we considered the dominant, recessive and additive genetic models (Table 2), and all were statistically significant (p 64 years old (n = 101) as shown in Table S2, but found no significant differences. Lack of OASL SNP rs3213545 association with symptomatic WNV disease We next attempted to validate the association previously identified by Yakub et al. between OASL SNP rs3213545 and WNV infection [22]. Contingency analysis (3×2) did not reveal any significant association (p = 0.93) when the WNV-infected seropositive subjects (n = 501) were compared to WNV-seronegative controls (n = 552). Analysis of the additive model (OR = 1.0 [95%CI 0.8–1.3], p = 0.94), the recessive model (OR = 1.0, [95%CI 0.69–1.6], p = 0.83), and the dominant model (OR = 1.0, [95%CI 0.8–1.3], p = 0.82) revealed no association with WNV infection (Table S3). When samples collected from each of the 5 centers were analyzed separately, no significant association was found (Table S3). The frequency of TT homozygosity was not elevated above controls (Figure 3B) and did not vary between asymptomatic donors and symptomatic patients. Since the previous study by Yakub et al. showed an association between this SNP and severe WNV disease, we also looked for an association according to clinical outcome. As shown in Table 3 and Figure 3B, no association was found with either WNF or WNND, the more severe CDC clinical definition. Development of an ex vivo model for WNV replication in human lymphoid tissue To directly assess the role of OAS1 during active WNV replication in human cells, we developed an experimental ex vivo model of WNV infection using explants of human tonsil tissue. This model may mimic the early events after infection that may occur in vivo in the draining lymph node, shown in mice to be an early site of viral replication that precedes CNS spread [30]. Since infection in primary human cells in vitro typically requires exogenous stimulation or activation [31],[32], this system is more natural in that the architecture and composition of the primary lymphoid tissue are preserved and no exogenous stimuli are added. Explanted tissue from patients undergoing tonsillectomies was sectioned and inoculated with WNV. For each experimental condition, 27 blocks of tissue were sectioned per donor to control for variation in the number of susceptible cells that may occur between individual slices of tissue from the same donor. Supernatant from the cultured tissue was tested every third day for virus using a focus forming unit (FFU) assay. As shown in Figure 4A, virus was detected in the culture supernatant at the earliest time point (day 3) and increased throughout the 12 day culture for each of the 21 donors tested. WNV-infected cells were visible by direct immunostaining of the tissues (Figure 4B). Tonsils from all donors supported WNV replication to high levels that varied widely among donors, particularly at early time points post-infection (e.g. greater than 4 logs variability at day 3). This model may be useful not only for investigating WNV pathogenesis but also for testing the efficacy of potential WNV therapeutics. 10.1371/journal.ppat.1000321.g004 Figure 4 Ex vivo model of WNV replication in cultured human lymphoid tissue. (A) Blocks of human tonsil tissue were inoculated ex vivo with WNV. Culture supernatant from WNV-infected and -uninfected tonsil tissue was sampled every third day for 12 days and viral titers assessed by FFU assay. 27 tissue blocks were used per experimental condition for each donor. The calculated maximal viral titer based on inoculum alone on day 0 is 1500 FFU/ml. Data represent the mean value of duplicate measurements with standard deviation. Note that data were not assessed for three patients on day 12. (B) Representative WNV immunostaining of WNV-infected (left panel) or donor-matched uninfected lymphoid tissue (right panel) cultured ex vivo for nine days. Scale bar, 100 µm. Early WNV replication may be regulated by OAS1 genotype The variation in virus production we observed among donors provided an ideal opportunity to test the dependence of viral replication on OAS1 genotype in intact human tissue, assuming that OAS1 mRNA was expressed in the tissue. Twelve of the 21 tissues tested for WNV replication were available for RNA extraction to investigate this. First we examined the effect of WNV infection on expression of all OAS1 transcripts (Figure 5A), using primers located between exons 2 and 3. OAS1 mRNA was detected in uninfected tissue from all 12 donors (data not shown), and expression of total OAS1 mRNA was induced ∼2 fold by WNV compared with donor-matched uninfected control tissue at day 3 post-infection (p = 0.01; Figure 5A). This increased to ∼4.6 fold induction for both day 6 (p = 0.002) and day 12 (p AG>AA, OAS enzymatic activity of purified protein encoded by OAS1 A and G allele variants has not yet been determined [23]. Furthermore, the mechanism of action of oas1b in mice is not yet clear, nor is there evidence that oas1b is enzymatically active [16] or that it functions through activation of RNaseL [15]. Population stratification as a cause of skewed genotypic frequency is a concern in all gene association studies. Given the limited availability of genomic DNA from the small samples of serum available to us from WNV+ subjects, we were unable to address this issue directly. However, we believe this is unlikely to confound our results since the five WNV+ groups were from geographically distinct US populations yet had similar AA genotypic frequencies ranging from 47.1% to 51.4% with an average of 49.3%, whereas the two US WNV-negative control populations that we genotyped, which also were from distinct geographic regions, had much lower AA genotypic frequencies (36.7 and 40.6%) that both fell within a narrow range reported in previously published studies, (36.8–41.1%) [27],[28],[29]. In particular, Smyth et al reported an OAS1 SNP rs10774671 AA genotypic frequency of 41.1% for 4,735 subjects from the 1958 British Birth Cohort (www.cls.ioe.ac.uk/studies.asp?section=000100020003)[29]; Fedetz et al reported a frequency of 36.8% for 424 Caucasians from Spain [27]; and the International HapMap Project reported a frequency of 39% in US Caucasians (n = 59; www.hapmap.org). It is important to note that a fourth published study, by Field et al., reported an AA genotypic frequency of 49.9% [28]. This anomaly is most likely due to the use of a non-random population, namely the healthy siblings of diabetics. In particular, the A allele frequency in non-transmitted alleles of the parents, which would be expected to have nothing to do with disease, was 0.61 in this study, which would give an expected frequency of 0.61×0.61 = 37% for the AA genotype (L. Field, personal communication), compatible with the frequency found in all five other control populations. Of note, no difference in frequency was observed between cases and controls in a genetically unlinked but related variant in OASL (www.hapmap.org), where the odds ratio for all genetic models tested was 1.0, p≥0.82. Animal studies have shown conclusively that the type I IFN system exerts potent antiviral effects critical for survival from viral infections, including WNV infection [9],[30],[33],[34]. However, there is limited evidence in humans supporting a role for endogenous type I IFN signaling at the level of viral pathogenesis. Our study suggests a potentially important role of this type I IFN-inducible pathway at the level of initial infection and containment of virus during the early phases of natural human infection. Our data show that WNV infection is less likely to result in seroconversion in individuals with the GG genotype (higher OAS enzymatic activity), suggesting that individuals with the AA genotype (lower enzymatic activity) are less likely to control initial WNV infection with innate immunity and initiate an adaptive immune response, signaled by seroconversion. Genetic variation in OAS1 has been previously associated with other diseases, including Type I Diabetes, SARS, and Hepatitis C. The associations in Type I Diabetes and SARS have been inconsistent in different studies [28],[29],[35],[36]. For Hepatitis C, a relative of WNV also in the flavivirus family, a polymorphism in the 3′-untranslated region of OAS1 has been associated with persistent infection [37]. This is particularly relevant since this polymorphism is in strong linkage disequilibrium with polymorphism rs10774671 which we have associated with WNV seroconversion [23]. A clinical implication of our results is that pharmacologic induction of OAS1, which has been shown to occur in response to IFNα treatment [38],[39] could be efficacious in the context of preventing WNV infection in man. While treatment with IFNα has been reported to be effective in WNV-infected patients, the world experience is limited to a small number of scattered case reports, and treatment failure has also been documented [40],[41],[42]. Our data support continued clinical research of IFNα as a therapeutic option in WNV disease. Finally, our data indicate that the great majority of risk of symptomatic WNV disease remains unexplained at the genetic level, and support continued research into variation in the type I IFN system as a factor contributing to the heterogeneity of outcome in this disease. Materials and Methods Study populations The study was approved by the Office of Human Subjects Research of the NIH. Samples of symptomatic WNV-seropositive patient cases from four US states (AZ, CO, CA, and IL) were collected as previously described [20],[21]. Since the allele frequency of this SNP varies by race (www.hapmap.org), and since information about race was not available for all subjects in the WNV-seropositive patient cases, we analyzed only self-reporting Caucasians (total n = 331 in the 4 US states combined). Symptomatic WNV-seropositive patients are defined as individuals who came to clinical attention with symptoms consistent with WNV disease (primarily WNF, WNND), where WNV was confirmed using serological tests. Of the WNV-seropositive samples received, sufficient DNA from self-reported Caucasian patients was available for analysis of 345 samples. Genotypes for both OAS1 and OASL were obtained for 331 symptomatic WNV-seropositive subjects with an overall genotyping success rate of 95.9%. Serum samples were collected from the following states and years: 1) Arizona from the 2004 epidemic (n = 135); 2) Colorado from the 2003 epidemic (n = 72); 3) California from the 2005 epidemic (n = 87); 4) Illinois from the 2005/2006 epidemic (n = 37). Plasma samples from asymptomatic WNV-seropositive blood donors from the American Red Cross (ARC WNV+) were collected between 2003–2008. Asymptomatic WNV-seropositive blood donors (n = 170) are defined as Caucasian random blood donors who tested reactive for WNV nucleic acid twice and were WNV IgM seropositive who remained asymptomatic for at least 2 weeks post donation as assessed by follow-up questionnaire as described previously [25]. Two healthy US Caucasian control groups were established: 1) healthy unrelated US Caucasian random blood donors (RBD) from the NIH Department of Transfusion Medicine (n = 360) collected under an IRB-approved protocol [20]; and 2) healthy Caucasian blood donors from the American Red Cross (n = 192) who tested WNV nucleic acid reactive upon initial screen at the time of blood donation, but were negative for WNV nucleic acid upon retesting and were also WNV-seronegative for IgM (false positives). The following information was provided if available: age, gender, self-reported racial group, date of sample collection, and CDC-defined clinical presentation at the time of sample collection: WNF, WNND, and death. For all study samples, investigators were blinded to unique patient identifiers. DNA isolation and genotyping 200 µl of serum collected from Arizona, Colorado, California, and Illinois were thawed for genomic DNA purification using the QiaAmp 96 DNA Blood Kit according to the manufacturer's instructions (Qiagen, Valencia, CA). 250 µl of plasma collected from test subjects by the American Red Cross was purified using NucliSENS EasyMAG automated nucleic acid extraction technology (Biomerieux, Inc). Purified DNA was eluted into 100 µl of the recommended buffer and stored at 4°C until further use. DNA from NIH random blood donors was isolated from peripheral blood leukocytes as previously described [43]. SNPs were genotyped using the ABI 7900HT PCR System with TaqMan primer/probe mix predesigned and validated by ABI (C___2567433_10 and C__11753831_1_). PCR-RFLP was used as a second genotyping method for OAS1 SNP rs10774671 as previously described [27]. Statistical analysis Contingency tables were utilized to calculate genotypic and allelic frequency differences between cases and controls by comparing numbers of expected (Hardy-Weinberg equation) and observed individuals using chi-square tests of significance to obtain a two-sided p value using either 1 (2×2 table) or 2 (3×2 table) degrees of freedom. ORs were calculated using a recessive genetic model (i.e. AA verses AG plus GG for OAS1 and TT versus TC plus CC for OASL) or the dominant genetic model (i.e. AA plus AG versus GG for OAS1 and TT plus TC versus CC for OASL) by cross tabulation and 95% CI were estimated using the approximation of Woolf (GraphPad Software version 4.0b, San Diego, CA). The additive model OR values and 95% CI were calculated using JMP software. Chi-square was used unless otherwise indicated. ORs and CIs based on the additive model were estimated using JMP (SAS, version 7). Chi-square goodness-of-fit statistics were used to compare dominant, recessive, and additive models. The two-sided unpaired t-test was used to calculate statistical significance of viral replication after log10 transformation of the data for each time point and a two-sided paired t-test was used to calculate significance of gene expression analysis. P values were considered significant if <0.05 and the reported values have not been corrected for multiple comparisons. Linkage disequilibrium of OAS1 SNPs and resultant haplotypes were examined using Haploview 4.0 (available at www.broad.mit.edu/mpg/haploview/index) using previously obtained data from the International HapMap project (www.hapmap.org) on the CEU panel using release 21 data. Infection of human lymphoid tissue ex vivo Human tonsils from routine tonsillectomy performed at the Children's Hospital National Medical Center in Washington, DC were collected under an Institutional Review Board-approved protocol from NICHD within a few hours after surgery. The mean age of the children used in this study was 5±3. Tonsils were dissected in blocks of approximately 2 mm. 9 tissue blocks per well in triplicate wells per condition were placed on top of collagen sponge gels floating in six-well plates as previously described [44]. All 27 tissue blocks from each donor were then individually inoculated with 5 µl containing 500 FFU WNV strain NY99, or left uninfected. We assessed productive WNV infection by measuring FFU as described above in culture medium pooled from 27 blocks during the previous 3 days between successive media changes. FFU at each time point represent the cumulative total of infectious virions produced. Coefficient of variation for each donor and each time point was calculated to be 13.3%. All tonsil donors were analyzed for the CCR5Δ32 mutation as previously described [20] and found to be wild type at this allele. Viral Titers Confluent Vero cells were grown in a 12 well plate in OptiPro SFM (Invitrogen) with 2% FBS (Hyclone) and 50 µg/ml gentamicin (ATCC). Supernatants from tonsil cultures were diluted and incubated on the cells for 1 hour at 37°C prior to overlaying with 2 ml Opti-MEM (Invitrogen) with 8 g/L methylcellulose (Sigma), 2% FBS and 50 µg/ml gentamicin. Plates were incubated for 2 days at 37°C, then washed 3 times with PBS. 500 µl of diluted anti-WNV anti-sera/well (HMAF, ATCC #VR-82) was incubated for 1 hour at 37°C. Plates were then washed 3 times and 500 µl of diluted (1∶10) anti-mouse/anti-rabbit HRP labeled polymer (DAKO Cytomation) was added. Cells were incubated for 1 hour at 37°C, washed 3 times and focus forming units (FFU) of WNV were visualized by addition of 1 ml diaminobenzidine (DAB) mixture (4.5 mg DAB (Sigma) /10 ml PBS+4.5 µl 30% H202 /10 ml). Viral titers are expressed as FFU/ml. Real-time PCR RNA was extracted from lymphoid tissue using the RNeasy Tissue kit according to manufacturer's protocol (Qiagen). Reverse transcription was performed using Superscript III first strand synthesis Supermix (Invitrogen) with random hexamers. OAS1 (Hs00242943_m1), IFNβ1 (Hs00277188_s1) and GAPDH (Hs00266705_g1) primer/probe sets were obtained from ABI and cycled on an ABI 7900HT PCR System. Each sample was normalized to GAPDH and fold change in infected samples were compared to uninfected donor-matched samples. Immunohistochemistry 4–5 µm sections of paraffin-embedded slides were stained with anti-WNV hyperimmune mouse ascites fluid (HMAF, ATCC #VR-82) diluted 1∶100 in antibody diluent, background reducing agent (DAKO Cytomation). After incubation with anti-mouse polymer-horse radish peroxidase (DAKO Cytomation), slides were developed with streptavidin and diaminobenzidine liquid (DAKO Cytomation) and counterstained with hematoxylin. Detection of OAS1 splice variants Tissues from 12 tonsil donor samples were tested for OAS1 SNP-specific mRNA. cDNA from uninfected and infected donor-matched samples for each time point amplified by PCR as previously described [23]. Briefly, primers were designed to flank the splice acceptor site (F-ggcggaccctacaggaaact; R-acaccagctcactgaggagc). The presence of a G allele resulted in a 416 bp amplicon, while the presence of the A allele resulted in 319 bp and 417 bp fragments. PCR products were resolved on a 2% agarose gel, visualized using ethidium bromide, and quantitated by densitometry normalized to β-actin using Image J software version 1.38×. Fold change was calculated by comparing normalized WNV-infected measurements to normalized uninfected measurements for each time point for each donor. Supporting Information Table S1 Distribution of OAS1 (rs10774671) and CCR5Δ32 genotypes in symptomatic WNV-seropositive patients (0.03 MB DOC) Click here for additional data file. Table S2 Distribution of OAS1 (rs10774671) genotypes according to age in Caucasian symptomatic WNV-seropositive patient samples (0.03 MB DOC) Click here for additional data file. Table S3 OAS-Like SNP rs3213545 is associated with increases risk of WNV infection (0.04 MB DOC) Click here for additional data file.
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Journal ID (iso-abbrev): Cardiology
Title:
Cardiology
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S. Karger AG
ISSN
(Electronic):
1421-9751
ISSN
(Print):
0008-6312
Publication date
(Electronic):
2008
Volume: 111
Issue: 3
Affiliations
Article
Publisher Item ID: 000121604DOI: 10.1159/000121604
PubMed ID: 18434725
SO-VID: b74f7552-6eb7-4933-8fff-071848c5e387
History
Data availability: